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Association of the N-formyl-Met-Leu-Phe receptor in human neutrophils with a GTP-binding protein sensitive to
Abstract:
Pertussis toxin inhibits the N-formyl-Met-Leu-Phe (fMet-Leu-Phe) mediated human neutrophil functions of enzyme release, superoxide generation, aggregation, and chemotaxis. As pertussis toxin modifies the GTP binding receptor-regulatory protein "Ni," the association of the fMet-Leu-Phe receptor with such a protein was further examined in purified neutrophil plasma membranes. Both fMet-Leu-Phe-mediated guanine nucleotide exchange and nucleotide-mediated regulation of the fMet-Leu-Phe receptor are inhibited by pertussis toxin. In addition, membrane pretreatment with pertussis toxin abolishes the fMet-Leu-Phe-mediated inhibition of adenylate cyclase. Actions of pertussis toxin are due to the ADP-ribosylation of a single subunit at 41 kDa in the neutrophil plasma membrane, which comigrates on NaDodSO4 gels with the Ni GTP-binding protein in the platelet plasma membrane. Our results suggest that (i) the fMet-Leu-Phe receptor is associated with a Ni GTP regulatory protein, and (ii) a fMet-Leu-Phe-Ni complex is important in the control of several neutrophil functions, probably involving multiple transduction systems, including adenylate cyclase.
Insights
Pertussis toxin blocks key human neutrophil functions by inhibiting the N-formyl-Met-Leu-Phe receptor. This toxin targets a GTP-binding protein, suggesting a crucial role in neutrophil signaling pathways.
Area of Science:
- Immunology
- Cellular Signaling
- Molecular Biology
Background:
- Human neutrophils initiate critical immune responses upon activation by N-formyl-Met-Leu-Phe (fMet-Leu-Phe).
- Pertussis toxin is known to interfere with cellular signaling pathways involving GTP-binding proteins.
Purpose of the Study:
- To investigate the mechanism by which pertussis toxin inhibits fMet-Leu-Phe mediated neutrophil functions.
- To examine the association between the fMet-Leu-Phe receptor and GTP-binding proteins in neutrophils.
Main Methods:
- Purification of human neutrophil plasma membranes.
- Assessing fMet-Leu-Phe receptor-mediated guanine nucleotide exchange and adenylate cyclase activity.
- Pertussis toxin treatment and analysis of protein modification via ADP-ribosylation.
Main Results:
- Pertussis toxin inhibited fMet-Leu-Phe mediated enzyme release, superoxide generation, aggregation, and chemotaxis.
- The toxin blocked fMet-Leu-Phe receptor-mediated guanine nucleotide exchange and adenylate cyclase inhibition.
- Pertussis toxin ADP-ribosylated a 41 kDa protein subunit in neutrophil membranes, consistent with the Ni GTP-binding protein.
Conclusions:
- The fMet-Leu-Phe receptor is associated with a Ni GTP regulatory protein in human neutrophils.
- A complex between the fMet-Leu-Phe receptor and Ni GTP-binding protein is vital for controlling neutrophil functions, likely through multiple signaling pathways including adenylate cyclase.